Clonorchis sinensis (C. sinensis) is one of the most serious food-borne parasites, which can lead to liver fibrosis or cholangiocarcinoma. Effective measures for clonorchiasis prevention are still urgently needed. Bacillus subtilis (B. subtilis) is an effective antigen delivery platform for oral vaccines. Chonorchis sinensis serpin (CsSerpin) was proved to be potential vaccine candidates. In this study, CsSerpin3 was displayed on the surface of B. subtilis spore and recombinant spores were orally administrated to BALB/C mice. CsSerpin3-specific IgA levels in faecal, bile and intestinal mucous increased at 4–8 weeks after the first administration compared with those in control groups. The mucus production and the number of goblet cells in intestinal mucosa elevated in B.s-CotC-CsSerpin3 (CotC, coat protein of B. subtilis spore) spores treated group compared to those in blank control. No significant difference in the activities of glutamic-pyruvic transaminase/ alanine aminotransferase and glutamic oxalacetic transaminase/aspartate aminotransferase were observed between groups. There was no side effect inflammation and observable pathological damage in the liver tissue of mice after administration. Moreover, collagen deposition and Ishak score were statistically reduced in B.s-CotC-CsSerpin3 spores treated mice. In conclusion, B. subtilis spores displaying CsSerpin3 could be investigated further as an oral vaccine against clonorchiasis.

This study evaluated the effect of Bacillus subtilis C-3102 (Calsporin®) addition to the diet on faecal characteristics and nutrient digestibility in healthy adult dogs. Sixteen Beagles received either a low-energy control diet (CON; 3.35 Mcal metabolisable energy (ME)/kg with 21.8, 27.9, and 50.3% ME as protein, fat, and nitrogen-free extractives (NFE), respectively) or the same diet supplemented with Bacillus subtilis at 1 × 109 CFU/kg diet as probiotic (PRO) for four weeks in a parallel design (eight dogs per diet). In the prior two weeks, all dogs received a high-energy diet (Advance Medium Adult, Affinity Petcare®, 3.81 Mcal ME/kg ME with 24.8, 41.2, and 34% ME protein, fat, and NFE, respectively). Faecal consistency, dry matter (DM), pH, and NH3 were analysed on fresh samples collected at the start and weekly throughout the study. Additional samples were collected for the determination of lactate and short-chain fatty acids (SCFA) on days 0 and 21. In week four, a five–day total faecal collection was conducted in six dogs from each diet for the determination of nutrient apparent digestibility. Dogs fed the PRO diet had more firm faeces (P = 0.011) than control dogs and a higher faecal DM content in the first two weeks (P < 0.05). Feeding the PRO diet resulted in a decline in NH3 over four weeks (P = 0.05) and in faecal pH in the first two weeks (P < 0.05) alongside an increase in SCFA content (P = 0.044), mainly acetate (P = 0.024). Faecal lactate did not differ between diets (P > 0.10). Dogs fed the PRO diet showed a higher apparent digestibility of fat (P = 0.031) and NFE (P = 0.038) compared to control dogs. Dog food supplementation with Calsporin® at 1 × 109 CFU/kg improved faecal quality, enhanced fat and carbohydrate digestibility, and contributed to the gut health of dogs by reducing gut ammonia and increasing SCFA content.

Whole-crop maize forage was ensiled without inoculant (control), inoculated with Lactobacillus buchneri and L. plantarum at a rate of 1 × 105 cfu/g fresh forage per bacterium (LBLP), or inoculated with Bacillus subtilis and L. plantarum at a rate 1 × 105 cfu/g fresh forage per bacterium (BSLP) with the goal to investigate the growth performance of finishing feedlot lambs. Thirty Dorper × Santa Ines lambs (29 ± 3.5 kg initial body weight) were used in the feedlot programme and assigned (n = 10) to one of three diets containing control, LBLP or BSLP silages in a 60:40 forage:concentrate ratio. Inoculation of maize silage did not alter dry matter intake (overall mean = 1.16 kg/day) and average daily gain (overall mean = 0.217 kg/day) of lambs. Consequently, feed efficiency remained unchanged. Inoculation of maize silage did not alter carcass and meat traits of lambs, with the exception of meat colour, wherein yellowness (b*) decreased by feeding LBLP and BSLP diets compared with the untreated diet. Regarding ruminal fermentation, there was an interaction between diets and the interval at which ruminal fluid was sampled for determining total volatile fatty acid concentration, but inoculation yielded no obvious results. In conclusion, the use of diets based on maize silage inoculated with L. plantarum combined with either L. buchneri or B. subtilis did not display relevant effects on growth performance of lambs; this response might be related to the limited impact of these bacterial inoculants on silage composition.

Mutants of Bacillus subtilis can be developed to overproduce Val in vitro. It was hypothesized that addition of Bacillus subtilis mutants to pig diets can be a strategy to supply the animal with Val. The objective was to investigate the effect of Bacillus subtilis mutants on growth performance and blood amino acid (AA) concentrations when fed to piglets. Experiment 1 included 18 pigs (15.0±1.1 kg) fed one of three diets containing either 0.63 or 0.69 standardized ileal digestible (SID) Val : Lys, or 0.63 SID Val : Lys supplemented with a Bacillus subtilis mutant (mutant 1). Blood samples were obtained 0.5 h before feeding and at 1, 2, 3, 4, 5 and 6 h after feeding and analyzed for AAs. In Experiment 2, 80 piglets (9.1±1.1 kg) were fed one of four diets containing 0.63 or 0.67 SID Val : Lys, or 0.63 SID Val : Lys supplemented with another Bacillus subtilis mutant (mutant 2) or its parent wild type. Average daily feed intake, daily weight gain and feed conversion ratio were measured on days 7, 14 and 21. On day 17, blood samples were taken and analyzed for AAs. On days 24 to 26, six pigs from each dietary treatment were fitted with a permanent jugular vein catheter, and blood samples were taken for AA analysis 0.5 h before feeding and at 1, 2, 3, 4, 5 and 6 h after feeding. In experiment 1, Bacillus subtilis mutant 1 tended (P<0.10) to increase the plasma levels of Val at 2 and 3 h post-feeding, but this was not confirmed in Experiment 2. In Experiment 2, Bacillus subtilis mutant 2 and the wild type did not result in a growth performance different from the negative and positive controls. In conclusion, results obtained with the mutant strains of Bacillus subtilis were not better than results obtained with the wild-type strain, and for both strains, the results were not different than the negative control.

In this work, fluorescent Bacillus subtilis (B. subtilis) cells were developed as probes for imaging applications and to explore behaviorial interaction between B. subtilis and Staphylococcus aureus (S. aureus). A novel biological strategy of coupling intracellular biochemical reactions for controllable biosynthesis of CdSe quantum dots by living B. subtilis cells was demonstrated, through which highly luminant and photostable fluorescent B. subtilis cells were achieved with good uniformity. With the help of the obtained fluorescent B. subtilis cells probes, S. aureus cells responded to co-cultured B. subtilis and to aggregate. The degree of aggregation was calculated and nonlinearly fitted to a polynomial model. Systematic investigations of their interactions implied that B. subtilis cells inhibit the growth of neighboring S. aureus cells, and this inhibition was affected by both the growth stage and the amount of surrounding B. subtilis cells. Compared to traditional methods of studying bacterial interaction between two species, such as solid culture medium colony observation and imaging mass spectrometry detection, the procedures were more simple, vivid, and photostable due to the efficient fluorescence intralabeling with less influence on the cells’ surface, which might provide a new paradigm for future visualization of microbial behavior.

The experiment SPORES ‘Spores in artificial meteorites’ was part of European Space Agency's EXPOSE-R mission, which exposed chemical and biological samples for nearly 2 years (March 10, 2009 to February 21, 2011) to outer space, when attached to the outside of the Russian Zvezda module of the International Space Station. The overall objective of the SPORES experiment was to address the question whether the meteorite material offers enough protection against the harsh environment of space for spores to survive a long-term journey in space by experimentally mimicking the hypothetical scenario of Lithopanspermia, which assumes interplanetary transfer of life via impact-ejected rocks. For this purpose, spores of Bacillus subtilis 168 were exposed to selected parameters of outer space (solar ultraviolet (UV) radiation at λ>110 or >200 nm, space vacuum, galactic cosmic radiation and temperature fluctuations) either as a pure spore monolayer or mixed with different concentrations of artificial meteorite powder. Total fluence of solar UV radiation (100–400 nm) during the mission was 859 MJ m−2. After retrieval the viability of the samples was analysed. A Mission Ground Reference program was performed in parallel to the flight experiment. The results of SPORES demonstrate the high inactivating potential of extraterrestrial UV radiation as one of the most harmful factors of space, especially UV at λ>110 nm. The UV-induced inactivation is mainly caused by photodamaging of the DNA, as documented by the identification of the spore photoproduct 5,6-dihydro-5(α-thyminyl)thymine. The data disclose the limits of Lithopanspermia for spores located in the upper layers of impact-ejected rocks due to access of harmful extraterrestrial solar UV radiation.

In this study, the effect of exposing entomopathogenic bacteria isolated from macerated termite cadavers to varying intensities of a magnetic field for different periods of time on their pathogenic potential was examined; pathogenicity tests were carried out for each of the bacterial species. Two of the bacteria, Bacillus subtilis (Ehrenberg) Cohn and Serratia marcescens Bizio, were able to induce morbid effects on termites and both were re-isolated from the resulting cadavers. Reinfection using different concentrations of both bacteria was carried out on termites to determine the minimum lethal concentration required for pathogenicity. Bacillus subtilis was able to degenerate the termites at concentration values of 108 colony-forming units (cfu)/ml and S. marcescens at 107cfu/ml. Both bacteria were then exposed to magnetic fields of different intensities for different periods of time, after which they were used for reinfection of healthy termites. Post-infection study after the exposure of termites to magnetic field-treated bacterial cells revealed no reduction in the entomopathogenic potency of S. marcescens. As the extensive use of chemicals to control insect pests has been found to have detrimental effects on people and the environment, there is a pressing need to discover and develop new entomopathogens to control these insects biologically. Therefore, bacteria discovered in this study to have entomopathogenic potency against termites may be further studied and formulated into either powdery forms or suspensions to be applied to infested wood or wood products.

Bacterial endospores are resistant to many environmental factors from temperature extremes to ultraviolet irradiation and are generally more difficult to inactivate or kill than vegetative bacterial cells. It is often considered necessary to treat spores or samples containing spores with chemical fixative solutions for prolonged periods of time (e.g., 1–21 days) to achieve fixation/inactivation to enable electron microscopy (EM) examination outside of containment laboratories. Prolonged exposure to chemical fixatives, however, can alter the ultrastructure of spores for EM analyses. This study was undertaken to determine the minimum amount of time required to inactivate/sterilize and fix spore preparations from several bacterial species using a universal fixative solution for EM that maintains the ultrastructural integrity of the spores. We show that a solution of 4% paraformaldehyde with 1% glutaraldehyde inactivated spore preparations of Bacillus anthracis, Bacillus cereus, Bacillus megaterium, Bacillus thuringiensis, and Clostridium perfringens in 30 min, and Bacillus subtilis in 240 min. These results suggest that this fixative solution can be used to inactivate and fix spores from several major groups of bacterial spore formers after 240 min, enabling the fixed preparations to be removed from biocontainment and safely analyzed by EM outside of biocontainment.

The aim of the present experiment was to examine the influence of feeding diets containing a probiotic containing three Bacillus subtilis strains or zinc bacitracin (BMD) on bird performance, intestinal mucosa-associated avian pathogenic Escherichia coli (APEC), intestinal morphology and litter water-soluble phosphorus (WSP) of broilers fed corn-based diets. Three treatments were employed, either a control diet; the control diet supplemented with probiotic at 7.5 × 104 colony forming units (cfu) /g of feed or the supplemented with BMD (50g/tonne feed). Broiler starter and finisher diets, based on corn and soybean meal, were formulated and fed ad libitum to seven pens of 50 male broilers per treatment from days 1 to 42. During the 1-35 d periods, feed intake and weight gain were not influenced (P > 0.05) by dietary treatments, but probiotic supplementation improved (P < 0.05) 35-d FCR compared to the control and BMD-supplemented diets (1.395, 1.447 and 1.484, respectively). During the 1-42d period, feed intake and weight gain were not influenced (P > 0.05) by dietary treatments. However, probiotic improved (P = 0.05) and BMD tended (P = 0.07) to improve FCR compared to control diet. Villus height and crypt depth in the duodenum and jejunum were not influenced (P > 0.05) by dietary treatment. The number of mucosa-associated APEC was not influenced (P > 0.05) by dietary treatments. Probiotic and BMD supplementation had no effect (P > 0.05) on litter total phosphorus. However, BMD reduced (P < 0.05) litter WSP compared to control and probiotic supplemented diet. In conclusion, probiotic supplementation had no effect on intestinal morphology or WSP but improved broiler performance and can be used as an alternative to antibiotic growth promoters.

Milk powder taken to Antarctica on Shackelton's British Antarctic Expedition in 1907 was produced in New Zealand by a roller drying process in the first factory in the world dedicated to this process. Thermophilic bacilli are the dominant contaminants of modern spray-dried milk powders and the 1907 milk powder allows a comparison to be made of contaminating strains in roller-dried and spray-dried powders. Samples of milk powder obtained from Shackelton's Hut at Cape Royds had low levels of thermophilic contamination (<500 cfu ml−1) but the two dominant strains (Bacillus licheniformis strain F and Bacillus subtilis) were typical of those found in spray-dried powders. Soil samples from the floor of the hut also contained these strains, whereas soils distant from the hut did not. Differences in the RAPD profiles of isolates from the milk powder and the soils suggest that contamination of the milk from the soil was unlikely. It is significant that the most commonly encountered contaminant strain in modern spray-dried milk (Anoxybacillus flavithermus strain C) was not detected in the 1907 sample.

Over 25 years ago, Pace and coworkers described an activity called RNase M5 in Bacillus subtilis cell extracts responsible for 5S ribosomal RNA maturation (Sogin & Pace, Nature, 1974, 252:598–600). Here we show that RNase M5 is encoded by a gene of previously unknown function that is highly conserved among the low G + C Gram-positive bacteria. We propose that the gene be named rnmV. The rnmV gene is nonessential. B. subtilis strains lacking RNase M5 do not make mature 5S rRNA, indicating that this process is not necessary for ribosome function. 5S rRNA precursors can, however, be found in both free and translating ribosomes. In contrast to RNase E, which cleaves the Escherichia coli 5S precursor in a single-stranded region, which is then trimmed to yield mature 5S RNA, RNase M5 cleaves the B. subtilis equivalent in a double-stranded region to yield mature 5S rRNA in one step. For the most part, eubacteria contain one or the other system for 5S rRNA production, with an imperfect division along Gram-negative and Gram-positive lines. A potential correlation between the presence of RNase E or RNase M5 and the single- or double-stranded nature of the predicted cleavage sites is explored.